AKT3逆转miR-22-3p/29a-3p对LX-2细胞活化的协同抑制作用  

AKT3 reverses the synergistic inhibition of miR-22-3p/29a-3p on LX-2 cell activation

作　　者：张荣花 张亚楠 韩向阳 崔笑妍 田晓丽[2] 章广玲 刘志勇 Zhang Ronghua;Zhang Yanan;Han Xiangyang;Cui Xiaoyan;Tian Xiaoli;Zhang Guangling;Liu Zhiyong(School of Basic Medical Sciences,North China University of Science and Technology,Hebei Key Laboratory for Chronic Diseases,Tangshan 063210;Paraplegia Sanatorium of Tangshan,Tangshan 063000;School of Clinical Medicine,North China University of Science and Technology,Hebei Key Laboratory of Medical-Industrial Integration Precision Medicine,Tangshan 063000;Health Science Center of North China University of Science and Technology,Tangshan 063210)

机构地区：[1]华北理工大学基础医学院,河北省慢性疾病基础医学重点实验室,唐山063210 [2]唐山市截瘫疗养院,唐山063000 [3]华北理工大学临床医学院,河北省医工融合精准医疗重点实验室,唐山063000 [4]华北理工大学医学部,唐山063210

出　　处：《安徽医科大学学报》2023年第7期1132-1139,共8页Acta Universitatis Medicinalis Anhui

基　　金：河北省自然科学基金(编号:H2021209026);河北省人力资源和社会保障厅项目(编号:C20210340);河北省省级科技计划(编号:213777115D);河北省财政厅项目(编号:冀财预复[2020]397号)。

摘　　要：目的探讨丝氨酸/苏氨酸激酶3(AKT3)能否逆转miR-22-3p/29a-3p对人肝星状细胞LX-2活化的协同抑制作用。方法利用TargetScan、Starbase、miRDB和DIANA预测miR-22-3p和miR-29a-3p的共同靶基因,对筛选出的靶基因进行KEGG、GO和蛋白-蛋白互作(PPI)分析;RNAhybrid分析候选靶基因与两个miRNAs结合的自由能,并通过双萤光素酶报告实验确定它们的靶向关系;采用CCK-8、Transwell和实时荧光定量PCR(qRT-PCR)法检测LX-2细胞增殖、迁移以及纤维化标志物的表达。结果miR-22-3p和miR-29a-3p的共同靶基因有24个,且它们富集在与肝纤维化相关的信号通路和生物学过程;候选靶基因AKT3与miR-22-3p和miR-29a-3p的结合自由能分析结合双萤光素酶实验结果提示AKT3是miR-22-3p和miR-29a-3p的共同靶基因;miR-22-3p和miR-29a-3p mimics能够单独或协同抑制LX-2细胞的增殖、迁移以及纤维化标志物α-平滑肌肌动蛋白(α-SMA)和I型胶原α1链(COL1A1)mRNA的表达(P<0.05),AKT3过表达后能够逆转miR-22-3p和miR-29a-3p mimics对LX-2细胞的上述协同抑制作用(P<0.05)。结论AKT3过表达可逆转miR-22-3p和miR-29a-3p对LX-2细胞活化的协同抑制作用。Objective To investigate whether serine/threonine kinase 3(AKT3)is able to reverse the synergistic inhibition of miR-22-3p/29a-3p on the activation of human hepatic stellate cell LX-2.Methods TargetScan,Starbase,miRDB and DIANA were used to predict the common target genes of miR-22-3p and miR-29a-3p,and the selected target genes were analyzed by Kyoto encyclopedia genes and genomes(KEEG),gene ontology(GO),protein-protein interaction(PPI).The binding free energy of candidate target genes with two miRNAs was analyzed by RNAhybrid,and their targeting relationship was determined by double luciferase reporting experiment.CCK-8,Transwell and qRT-PCR were used to detect the proliferation,migration and the expression of fibrosis markers of LX-2 cells.Results There were 24 common target genes of miR-22-3p and miR-29a-3p,and they were enriched in the hepatic fibrosis-related signaling pathways and biological processes.The binding free energy analysis of candidate target gene AKT3 with miR-22-3p and miR-29a-3p combined with the double luciferase experiment results showed that AKT3 was the common target gene of miR-22-3p and miR-29a-3p.The proliferation,migration and the mRNA expression of fibrosis markers of α-smooth muscle actin(α-SMA)and type I collagenαl chain(COL1A1)of LX-2 cells were inhibited by miR-22-3p and miR-29a-3p mimics independently or cooperatively(P<0.05),and AKT3 overexpression could reverse the synergistic inhibition of miR-22-3p and miR-29a-3p mimics on LX-2 cells mentioned-above(P<0.05).Conclusion The overexpression of AKT3 can reverse the synergistic inhibition of miR-22-3p and miR-29a-3p on the activation of LX-2 cells.

关 键 词：LX-2 丝氨酸/苏氨酸激酶3 miR-22-3p miR-29a-3p 增殖 迁移 

分 类 号：R575.2[医药卫生—消化系统]